Review of Hair Follicle Dermal Papilla cells as in vitro screening model for hair growth

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1 International Journal of Cosmetic Science, 2018, 40, doi: /ics Review Article Review of Hair Follicle Dermal Papilla cells as in vitro screening model for hair growth Alka Madaan, Ritu Verma, Anu T. Singh and Manu Jaggi Cell Biology Lab, Dabur Research Foundation, 22, Site IV, Sahibabad, Ghaziabad, Uttar Pradesh , India Received 29 June 2018, Accepted 20 August 2018 Keywords: Dermal papilla cells, Hair growth, Hirsutism, In vitro, Screening model Abstract Hair disorders such as hair loss (alopecia) and androgen dependent, excessive hair growth (hirsutism, hypertrichosis) may impact the social and psychological well-being of an individual. Recent advances in understanding the biology of hair have accelerated the research and development of novel therapeutic and cosmetic hair growth agents. Preclinical models aid in dermocosmetic efficacy testing and claim substantiation of hair growth modulators. The in vitro models to investigate hair growth utilize the hair follicle Dermal Papilla cells (DPCs), specialized mesenchymal cells located at the base of hair follicle that play essential roles in hair follicular morphogenesis and postnatal hair growth cycles. In this review, we have compiled and discussed the extensively reported literature citing DPCs as in vitro model to study hair growth promoting and inhibitory effects. A variety of agents such as herbal and natural extracts, growth factors and cytokines, platelet-rich plasma, placental extract, stem cells and conditioned medium, peptides, hormones, lipid-nanocarrier, light, electrical and electromagnetic field stimulation, androgens and their analogs, stress-serum and chemotherapeutic agents etc. have been examined for their hair growth modulating effects in DPCs. Effects on DPCs activity were determined from untreated (basal) or stress induced levels. Cell proliferation, apoptosis and secretion of growth factors were included as primary end-point markers. Effects on a wide range of biomolecules and mechanistic pathways that play key role in the biology of hair growth were also investigated. This consolidated and comprehensive review summarizes the up-to-date information and understanding regarding DPCs based screening models for hair growth and may be helpful for researchers to select the appropriate assay system and biomarkers. This review highlights the pivotal role of DPCs in the forefront of hair research as screening platforms by providing insights into mechanistic action at cellular level, which may further direct the development of novel hair growth modulators. Resume Les problemes capillaires, tels que la perte de cheveux (alopecie) et la croissance excessive des cheveux dependante des androgenes (hirsutisme, hypertrichose), peuvent avoir un impact sur le bien-^etre social et psychologique de la personne. De recentes avancees dans la Correspondence: Alka Madaan, Cell Biology Lab, Dabur Research Foundation, 22, Site IV, Sahibabad, Ghaziabad, Uttar Pradesh , India. Tel.: +91- (0120) ; fax: +91- (0120) ; s: alka.madaan2012@gmail.com, alka.madaan@daburresearch.in connaissance de la biologie des cheveux ont accelere la recherche et le developpement de nouveaux agents therapeutiques et cosmetiques pour la croissance des cheveux. Les modeles precliniques contribuent aux analyses d efficacite dermo-cosmetiques et a la justification des allegations de modulateurs de croissance capillaire. Les modeles in vitro visant a etudier la croissance des cheveux utilisent les cellules du derme papillaire (Dermal Papilla Cells, DPC) du follicule pileux, des cellules mesenchymateuses specialisees situees a la base du follicule pileux, qui jouent un r^ole essentiel dans la morphogenese du follicule pileux et les cycles de croissance capillaire post-natals. Dans cette revue, nous avons compile et examine la vaste litterature rapportee, mentionnant les DPC comme modele in vitro pour etudier les effets stimulateur et inhibiteur de la croissance capillaire. Divers agents, tels que les extraits naturels et des extraits de plantes, les facteurs de croissance et les cytokines, le plasma riche en plaquettes, l extrait de placenta, les cellules souches et le milieu conditionne, les peptides, les hormones, les nanovecteurs lipidiques, la lumiere, la stimulation du champ electrique et electromagnetique, les androgenes et leurs analogues, les serums antistress et les agents chimiotherapeutiques, entre autres, ont ete examines pour leurs effets modulateurs de la croissance capillaire au niveau des DPC. Les effets sur l activite des DPC ont ete determines a partir de niveaux non traites (basiques) ou induits par le stress. La proliferation cellulaire, l apoptose et la secretion de facteurs de croissance ont ete incluses comme marqueurs du critere d evaluation primaire. Les effets sur un large eventail de biomolecules et voies mecanistiques jouant un r^ole cle dans la biologie de la croissance des cheveux ont egalement ete etudies. Cette revue exhaustive et approfondie resume les informations et connaissances actualisees concernant les modeles de selection bases sur les DPC pour la croissance des cheveux, et peut permettre aux chercheurs de selectionner le systeme de test et les biomarqueurs appropries. Cette revue met en evidence le r^ole fondamental des DPC au premier plan de la recherche capillaire comme plateformes de selection, en fournissant un apercßu du mecanisme au niveau cellulaire, ce qui peut orienter encore davantage le developpement de nouveaux modulateurs de croissance capillaire. Introduction Hair is a specialized derivative structure of skin and a defining characteristic of the integumentary system. Hair has a remarkable impact on social interactions and psychological well-being of 2018 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie 429

2 humans and plays an important role in regulating our body temperature [1]. The turnover of hair follicles (HF) occurs in cycles with alternating stages of rapid growth and hair shaft formation (anagen), apoptosis-driven regression (catagen) and relative HF quiescence (telogen). A disturbance in normal functioning of hair cycle results in various hair disorders. Alopecia (hair loss) is exceedingly prevalent condition which affects men and women of all ages. Only two drugs, finasteride and minoxidil have been approved for the treatment of hair loss by the US Food and Drug Administration (FDA) [2]. However, due to their limited efficacy and side effects, there has been a dramatic progress in the development of novel hair growth promoters [3]. On the other hand, excessive terminal hair growth in androgen-dependent areas in women with a typical male distribution pattern is known as Hirsutism [4]. Currently, anti-androgens, Gonadotropin-releasing hormone (GnRH) agonist, insulin-lowering drugs and Eflornithine hydrochloride are used to manage hirsutism. Due to the limitations associated with standard hair care products, such as limited and transient efficacy, unpredictable efficiency and side effects, there is an unmet need to design and develop novel and effective hair growth agents. The demand of therapeutically efficacious and safe agents has driven the search for novel hair growth modulators. Hence, there exists a need of developing suitable preclinical screening models to accelerate the research in this area. Various preclinical models have been developed by researchers to investigate the function of the hair follicle and understand the mechanism of action of compounds in hair growth [5]. Animal models have been used in hair research to gain a better understanding of the pathophysiological processes involved in hair loss and regeneration. The in vivo research on hair growth has been conducted so far on mice, rats, hamsters, rabbits and sheep in laboratory conditions. The screening models routinely employed for assessment of hair growth are in vivo C57BL/6 and C3H mice model and ex vivo human and mouse hair follicle cultures. Induction of anagen in alopecia model of C57BL/6 mice and hair shaft elongation are the primary end points in these models [6]. The root of hair is buried inside the epidermis and enclosed within a hair follicle (HF). The HF consists of the epithelial components (matrix and outer-root sheath) and dermal components (dermal papilla at the base of hair follicle and connective tissue sheath) [7]. Dermal papilla cells (DPCs) are specialized mesenchymal component of hair that play crucial role in morphogenesis and regeneration of hair growth. DPCs regulate the HF development and growth by acting as a reservoir of multi-potent stem cells, nutrients and growth factors [8]. Recently, DPCs have emerged as the focus of intense interest for researchers in hair biology. DPCs serve as excellent model to investigate the mechanistic action of compounds on functioning of human follicles. Owing to their important role in hair growth, DPCs have been classically used in numerous studies as in vitro screening model to evaluate the effect of hair growth modulating agents at cellular and molecular level. In addition, Outer Root Sheath (ORS) keratinocytes cultures were also employed as secondary in vitro screening model to understand the hair growth properties [9]. This was followed by assessment of the efficacy of compounds in ex vivo and in vivo models and correlation of their hair growth potential. The objective of this review is to systematically compile and discuss the key data available from the articles (published from 1987 to 2018) employing DPCs as in vitro model to evaluate various classes of compounds for hair growth promoting or inhibitory potential. Strategy for literature search The PubMed, Scopus and Google Scholar databases were searched for articles published from 1987 to the present. Search terms included in vitro hair growth and DPC, in vitro stimulatory/inhibitory effect and DPC, and DPCs and protective effect against stress. References from reviews about DPCs and molecular mechanism of hair growth were searched for additional articles. A manual search was also conducted based on citations in the published literature. Inclusion and exclusion criteria Selection criteria included articles that investigated the hair growth promoting or inhibitory effects of agents using cultured DPCs in vitro, via modulation of multiple mechanistic markers. Studies focusing solely on in vivo and ex vivo hair growth in animals with no experiments conducted on DPCs were excluded. Methodology of DPCs based models Cell culture The studies to examine hair growth modulating effects of compounds involved both primary cultures of DPCs isolated from human scalp follicles, mouse or rat vibrissae follicles, macaque and mink skin tissues. However, due to limited lifespan of primary cultures and large cell numbers required for setting up the assays, immortalized DPC lines have been developed by introducing human telomerase reverse transcriptase (htert) gene and are being extensively used in hair growth studies [10,11]. Primary and immortalized DPCs are also commercially available, accelerating the hair growth research. Test models The modulating effects of hair growth promoters on DPCs activity have been investigated by observing the changes in markers in either the basal model, where the changes are observed in comparison with untreated DPC activity, or the stress induced model where the protective effect of compounds is determined against conditions such as oxidative damage by H 2 O 2, UVB damage, catagen induction (Dexamethasone, IFN-c, DHT), cortisol and cisplatin etc. Inhibitory effects on DPCs activity have been evaluated in the basal model. End points The end points for hair growth comprised of cell proliferation, apoptosis and other mechanistic markers playing crucial role in the biology of hair growth. Proliferation is the most widely tested marker of DPCs activity. Proliferation of DPCs determines their growth rate and mitotic index; hence the resultant effect on cell proliferation suggests the hair growth promoting or inhibitory effects. Effect on apoptosis has been investigated using a range of early, mid and late apoptotic markers (annexin, caspase, ROS, PARP, cell cycle, Bcl-2/Bax). DPCs produce a plethora of cytokines, growth factors and other biomolecules that play a key role in hair biology [12]. Primarily, the effects on key cytokines such as VEGF, KGF, IGF, HGF, FGF, and TGF-b2 etc and their receptors have been determined. The Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie

3 modulation of mechanistic markers such as ALP, Wnt/b catenin pathway, JNK, ERK, Akt and MAPK etc indicated the effect on signaling pathways operating in DPCs. Migration potential using scratch based assay and 5-a reductase activity were other important markers. Markers of stemness such as Oct-4, Nanog, Sox2, CD133 etc were also tested. constituents as hair growth promoters has been driven by their less toxicity, easy availability, low-cost, patient compliance and more than one mode of biochemical action [29 31]. Specific effects of herbal extracts, polyherbal formulations, flavonoids and natural compounds on DPCs have been examined [32 108]. In vivo hair growth promoting effects as proof of concept are also mentioned. Hair growth promoting effect Minoxidil Minoxidil (2,4-diamino-6-piperidinipyrimidine-3-oxyde) is an antihypertensive vasodilator medication, used topically to treat hair loss in men and women [13, 14]. Introduced in 1970 as an anti-hypertensive agent, an increase in hair growth (hypertrichosis) was observed as a side effect of minoxidil [15]. Table I represents the studies wherein hair growth promotion mechanism of minoxidil was studied in DPCs [16 28]. Being the first line topical therapy for alopecia and due to the cellular mechanism determined in DPCs, minoxidil has been routinely used as a positive control in studies investigating the hair growth promoting effects of compounds. Table II summarizes the effects of various classes of agents on DPCs in vitro with the biomarkers evaluated. Herbal extracts, plant actives and natural products For centuries, herbs have been used to treat hair fall and promote hair growth worldwide. The research on herbs and active Table I Effects of Minoxidil on DPCs Cells Mechanism of action in DPCs Reference Human DPC PG6KF1a ( ) [16] PGE2 and LTB4 against A23187 ( ) Human and DNA synthesis by 3H-thymidine incorporation ( ) [17] macaque DPC Human DPC Modulation of collagen and arachidonic acid, [18,19] VEGF ( ) Human DPC Cytoprotective isoform of PGHS-1, PGE2 ( ) [20] Human DPC 17b-hydroxysteroid dehydrogenase, [21] 5a-Reductase activity ( ) Human DPC Opening of ATP-K channels ( ) [22] Intracellular Ca(2+) and VEGF ( ) Human DPC Proliferation by MTT ( ) [23] Bcl-2/Bax ratio, phosphorylation of ERK and Akt ( ) Human DPC b-catenin activity ( ) [24] Phosphorylation of GSK3b, PKA, PKB, Axin2, LEF-1, EP2 ( ) Human DPC HIF-1a and VEGF ( ) [25] Mouse DPC Apoptosis ( ) [26] Kras, ERK, Akt, SHH ( ) Human DPC Proliferation by MTT ( ) [27] Bcl-2/Bax ratio, phosphorylated ERK and Akt ( ) Human DPC Proliferation by MTT( ) Phosphorylated ERK and Akt, Bcl-2/Bax ratio ( ) P53, P21( ) [28] ( ) Stimulation, ( ) Inhibition. Growth factors and cytokines Various growth factors and cytokines are secreted from DPCs, which act as potent mitogenic factors and play important roles in follicular development and vasculogenesis. The resultant effects of growth factors and cytokines on proliferation of cultured DPCs and other mechanistic arms have been studied by researchers [ ]. Platelet rich plasma and placental extract Platelet-Rich Plasma (PRP) is an autologous preparation that concentrates platelets in a small volume of plasma. PRP has attracted significant attention of researchers in several medical fields such as cosmetics and dermatology due to wound healing potential. Treatment of follicular units with PRP resulted in significant improvement in hair density and stimulation of hair growth during male pattern baldness surgery. This may be mediated via release of growth factors from platelets that stimulate development of new follicles by promoting neovascularization [123]. Human placental extract (HPE) possesses Corticotropin-Releasing Factor (CRF)-like action, improvement of liver function and anti-platelet aggregation. PRP and HPE have demonstrated stimulatory effect on the activity of DPCs in vitro [26, ]. Stem cells and conditioned medium Owing to the role of stem cells in tissue regeneration, their beneficial effects in hair regeneration have recently emerged as a niche area of research. The conditioned medium secreted by stem cells also act as a highly enriched source of growth factors that stimulates hair growth. The hair growth promoting effects of stem cells and conditioned medium have been mediated via activation of DPCs [ ]. Peptide/proteins, squarticles and mimetics Several peptide complexes, proteins and mimetics have also displayed significant hair growth potential in DPCs [ ]. Squarticles are the nanoparticles formed from sebum-derived lipids such as squalene and fatty esters to achieve targeted drug delivery to the follicles. Their direct effects on DPCs have been studied in vitro [147, 148]. Light, electromagnetic field and electrical stimulation Low Level Laser Light Therapy (LLLT) is widely used as alternative medical treatment in wound healing and to relieve pain and inflammation. The effects of light therapy on hair growth have been investigated via DPCs [ ]. In addition, electromagnetic field resulted in stimulation of DPCs activity [152, 153]. Electrical stimulation also induced hair regrowth by inducing hair follicular proliferation and stimulation of DPCs in vitro [154, 155] Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie 431

4 Table II Stimulatory effects of various agents on DPCs Compound Cells Mechanism of action in DPCs In vivo/ex vivo activity Reference Herbal extracts Sophora flavescens extract Human DPC No increase in proliferation by [3H]thymidine incorporation IGF-1, KGF ( ) Hair Essence (HHRHG ) containing Biotae semen, Cornifructus, Coicis semen, Lycii fruictus, Glycyrrhizae radix Topically applied, Telogen to anagen conversion in C57BL/6 mice Rat prostatic type II 5a-reductase activity ( ) Rat vibrissae DPC Proliferation by MTT ( ) Topically applied, anagen induction and hair restoration in C57BL/ Asiasari radix extract Human DPC Proliferation by [3H]thymidine incorporation ( ) VEGF ( ) 6 mice Type II 5a reductase activity in HEK293 cell line ( ) Topically applied, Telogen to anagen conversion in C57BL/6 and C3H mice Protein synthesis in murine vibrissae follicles ( ) Sanguisorba Officinalis extract Rat Whisker DPC Proliferation by CCK-8 ( ) Topically applied, elongation of the anagen period in C3H mice, follicle length ( ) Erica multiflora extract Human DPC Proliferation by MTT ( ) G2/M phase in cell cycle ( ) Schisandra nigra extract Immortalized vibrissae DPC Norgalanthamine, a component of Crinum asiaticum Immortalized rat vibrissae DPC Fructus panax (Ginseng extract) Human DPC Proliferation by MTT ( ) Bcl-2/Bax ratio ( ) Black soybean (Glycine max: BB) Aconiti Ciliare Tuber extract Immortalized human and rat vibrissae DPC Acanthopanax koreanum extract Immortalized rat vibrissae DPC Grateloupia elliptica Immortalized rat vibrissae DPC Medicinal Herbal Complex Extract (mix of 23 herbs) Cepharanthine (alkaloid extracted from Stephania cepharantha) Polygonum multiflorum extract Immortalized rat vibrissae DPC Subcutaneously injected, Telogen to anagen induction in C3H/ HeN mice Proliferation ( ) Topically applied, anagen progression of the hair shaft in C57BL/6 mice TGF-b2 expression in vibrissae follicles ( ) Proliferation ( ) Topically applied, anagen progression in C57BL/6 mice Hair-fiber length and PCNA expression of rat vibrissa follicles ( ) [32] [33] [34] [35] [36] [37] [38] Topically applied, elongation of anagen phase in C57BL/6 mice [39] Rat vibrissae DPC Proliferation by MTT ( ) Topically applied, anagen induction and hair restoration in C57BL/ 6 mice Proliferation by WST-1 ( ) ALP activity ( ) Proliferation ( ) Nuclear b-catenin level, cyclin D1, cyclin E, CDK2 ( ) p27(kip1)( ) [40] Topically applied, induction of anagen hair growth in C57BL/6 [41] Hair-fiber lengths of rat vibrissae follicles ( ) [42] Proliferation by WST-1 ( ) Rat prostatic 5a-reductase activity ( ) [43] Human DPC Proliferation by MTT ( ) Topically applied, telogen to anagen transformation in C57BL/6 mice Mouse DPC Proliferation ( ) IGF-1 ( ) Crataegus pinnatifida extract Human DPC Proliferation ( ) ERK, p-38, JNK, Akt ( ) Origanum vulgare extract Human DPC No cytotoxicity or increase in proliferation by sulforhodamine (SRB) assay ( ) *Wheat Bran (n-hexane fraction) Immortalized rat vibrissae DPC Carthamus tinctorius floret extract Topically applied, induction of hair growth in C3H/HeN mice [45, 46] Proliferation by MTT ( ) Hair-fiber length of vibrissae follicles ( ) [47] Proliferation by MTT ( ) Cell cycle proteins-cyclin D1, cyclin E, phospho- prb ( ) Wnt/b-catenin signaling phospho(ser675)-b-catenin, phospho(ser9)-gsk3b ( ) Human DPC Proliferation by MTT ( ) VEGF, KGF ( ) TGF-b1 ( ) Orally administered, Anagen induction in C57BL/6 mice, Bcl-2/ Bax in skin ( ) [44] [48] Topically applied, hair growth in C57BL/6 mice, IGF-1 in skin ( ) [49] Topically applied, telogen-anagen transition in C57BL/6 mice Hair-fiber lengths of rat vibrissae follicles ( ) Topically applied, stimulated the growth of hair with local effects in C57BL/6 mice Length of cultured hair follicles ( ) [50] [51] Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie

5 Table II (continued) Compound Cells Mechanism of action in DPCs In vivo/ex vivo activity Reference CARI ONE (mixture of medicinal plants and mushrooms) Avicennia marina extract (Avicequinone C) Platycarya strobilacea S. et Z. Extract *Plant mixture (Glycyrrhiza uralensis, Angelica gigas, Acorus calamus, Cnidium officinale, Panax ginseng, Camellia sinensis, Salvia miltiorrhiza, Zanthoxylum schinifolium, Carthamus tinctorius, Prunus persica, Scrophularia buergeriana) Herbal extract Mixture (Chamaemelum Nobile, Althaea Officinalis, Persea Americana, Rosmarinus Officinalis, Aloevera, Urticadioica, Thymusvulgaris) Allium tuberosum Rottler ex Spreng extract Human DPC Proliferation by MTT ( ) Topically applied, stimulation of the telogen to anagen transition in C57BL/6 mice Number and size of hair follicles (HFs), earlier induction of b- catenin and Trp-1 protein ( ) Human DPC 5a-reductase type-i by TLC ( ) [53, 54] Human DPC Proliferation by CCK-8 ( ) Topically applied, transition from telogen to anagen in C57BL/6 mice Hair follicle length, SCF, IGF-1, KGF ( ) Mast cell number, TGF-b1( ) Human DPC Proliferation by MTT ( ) IGF-1, VEGF, KGF, HGF ( ) Human DPC Proliferation by MTT and BrdU ( ) CyclinD1, Cdk4, p-erk, p-akt, Bcl2/Bax ( ) 5a-Reductase2 activity ( ) Topically applied, early onset and prolongation of the anagen phase in C57BL/6J mice IGF-1, VEGF, KGF, HGF in skin ( ) 5a-reductase activity in rat microsomes ( ) [52] [55] [56] [57] Human DPC No increase in proliferation by MTT Topically applied, enhanced hair growth in telogenic C57/BL6 mice Number of hair follicles and IGF-1 in skin ( ) Ginsenoside Rg3 Human DPC VEGF ( ) CD8 and CD34 in mouse hair follicles ( ) [59] [60] Malva verticillata seed extracts (Myristoleic acid) Human DPC Wnt/b-catenin reporter activity ( ) Proliferation ( ) IGF-1, KGF, VEGF, HGF ( ) phosphorylation of MAPKs (Akt, p38) ( ) Artepillin C Human DPC Proliferation ( ) [61] [62] YangHyulEumGami-Bang (YHEG) 14 plants mix Glycine soja Siebold et Zucc Immortalized rat vibrissa DPC Human DPC Proliferation by [3H]thymidine incorporation ( ) IGF-1, KGF-1, b-catenin ( ) TGF-b1, BMP-2, NO ( ) Rumex japonicas Houtt extract Human DPC Proliferation by MTT ( ) Akt, ERK1/2, p-gsk3b, Wnt/b-Catenin ( ) Bcl-2/Bax ( ) *Geranium sibiricum extract Human DPC Proliferation by CCK-8 ( ) Cell migration ( ) Ki-67 protein, HGF, VEGF ( ) Topically applied, increased the hair growth index, number, length and size of the hair follicle 5-a reductase type II activity in HEK293 cell line ( ) Proliferation by MTT ( ) Topically applied, enhanced hair growth in C58BL/6 mice [63] Topically applied, induction of anagenic phase in telogenic C57BL/6 mice, Ki-67 and b-catenin ( ) Topically applied, induction of active anagen phase and SCF in C57BL/6 mice Mast cells and TGF-b1 in mouse skin ( ) Alpinia zerumbet Human DPC Proliferation by MTT ( ) [66] [67] Miscanthus sinensis var. purpurascens flower extract Human DPC TGF-b1 ( ) HGF, b-catenin ( ) Topically applied, telogen to anagen phase transition in C57BL/6 mice, Mast cells, Substance P and TGF-b ( ) SCF ( ) [58] [64] [65] 2018 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie 433

6 Table II (continued) Compound Cells Mechanism of action in DPCs In vivo/ex vivo activity Reference Yangxue Shengfa capsule (Chinese Proprietary Medicine) Immortalized human DPC Centella asiatica 3-D spheroid human DPC Proliferation MTT ( ) 5a-reductase activity by LC-MS/MS ( ) Modulation of IGF-1, DKK-1, TGF-b1 Centella asiatica Linn Human DPC Proliferation by MTT ( ) VEGF ( ) DA-5512 (Proprietary Herbal Extract of 6 oriental plants) Human DPC Proliferation MTT ( ) Ki-67-positive cells ( ) Proliferation by WST-1( ) STAT-targeted genes, SOCS1, SOCS3, p-stat ( ) ALPL, Versican, BMP2, NOG ( ) *Biota orientalis L. Extract Human DPC Proliferation of keratinocytes and DPC cocultures by CCK-8 ( ) KGF, VEGF ( ) *Lychee Fruit Polyphenol (Oligonol â ) *Cinnamomum osmophloeum Kanehira Leaf Aqueous Extract Flavonoids trans-3,4 0 -Dimethyl-3- hydroxyflavanone (t-flavanone) Human DPC No effect on proliferation by MTT FGF-7, Wnt10a ( ) IL-1b, IL-6, IL-8, IL-11,IL18r1, JUNB, FOS-like1, ICAM1, LAIr1, PTGS2, TRAF1 ( ) Rat vibrissae and human DPC Co-cultures of human DPC and human keratinocytes Proliferation by MTT ( ) HGF, VEGF, KGF, TGF-b2 ( ) Active TGF-b2 ( ) urokinase-type plasminogen activator (upa) activity ( ) [68] [69] [70] Topically applied, number and length of hair follicles in C57BL/6 mice ( ) [71] [72] [73] Topically applied, promotion of anagen phase in C57BL/6 mice [74] [75] Baicalin Human DPC Wnt/b-catenin signaling, ALP, IGF-1, VEGF ( ) Topically applied, earlier conversion from telogen to anagen in C57BL/6 mice Icariin Human DPC IGF-1 ( ) Orally administered, telogen to anagen transition in C57BL/6 mice, Hair shaft elongation, prolongation of anagen in cultured vibrissae hair follicles, IGF-1 ( ) Naringenin, Hesperetin Immortalized human DPC Green Tea Epigallocatechin-3- Gallate (EGCG) Emblica (Phyllanthus emblica Linn.) Human DPC Rat vibrissae Proliferation bymtt ( ) Cell survival against H2O2 ( ) VEGF ( ) Proliferation bymtt ( ) phosphorylated ERK, Akt ( ) Bcl-2/Bax ratio ( ) Cell cycle (S phase) ( ) cyclin B1 and cyclin D1 ( ) Shh, Akt ( ) [76] [77] [78] [79, 80] Human DPC Proliferation by MTT ( ) [81] L-Ascorbic acid-2-phosphate Human DPC Versican (V0 and V1), phosphorylated PKB, Nuclear beta-catenin ( ) Ascorbic acid-2-phosphate Human DPC IGF-1 ( ) CCN1/Cyr61 and CCN2/CTGF ( ) Nuclear accumulation of b-catenin ( ) Elongation of hair shafts in cultured human hair follicles, hair growth in mice [82, 83] IGF-1 in cultured hair follicles ( ) [84,85] Natural compounds Extracts from woods and fungi Rat vibrissae DPC Proliferation by WST-1 ( ) [86] Caffeine Human DPC Proliferation by MTT ( ) Proliferation of human hair follicles ( ) [87, 88] Apoptosis ( ) Modulation of AR, SRD5A2, p53, FasR, GSK-3b, TGFb2 genes Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie

7 Table II (continued) Compound Cells Mechanism of action in DPCs In vivo/ex vivo activity Reference *Ishige sinicola extract Immortalized Rat vibrissae DPC Combination of zeaxanthin, spermidine and rutin Proliferation-MTT ( ) phospho-gsk3b, b-catenin, Cyclin E, CDK2 ( ) p27(kip1) ( ) Human DPC Caspase-3 activity induced by Straurosporine ( ) Preserved Bcl2, Akt, MAPK Ecklonia cava Human DPC Proliferation by MTT ( ) IGF-1 ( ) Ecklonia cava Polyphenols Human DPC Proliferation by MTT, IGF-1, VEGF ( ) ROS ( ) Dieckol, a component of Ecklonia cava 7-Phloroeckol isolated from Ecklonia cava Immortalized rat vibrissae DPC Topically applied, promotion of anagen progression of the hair shaft in C57BL/6 mice Hair-fiber length in cultured vibrissae follicles ( ) Rat prostatic 5a-reductase activity ( ) [89] [90] Topically applied, induction of telogen to the anagen phase in the dorsal skin of C57BL/6 mice Elongation of the hair shaft in cultured human hair follicles ( ) [91] Elongation of human hair shaft length ex vivo ( ) [92] Proliferation by MTT ( ) Topically applied, anagen progression in C57BL/6 mice Hair-fiber length in cultured rat vibrissa follicles ( ) Rat prostatic 5a-reductase activity ( ) Human DPC MTT ( ) IGF-1 mrna, PI3K, p-akt, MEK½, phosphorylated ERK, c-fos, Bcl-2/Bax ratio( ) p53, caspase3/caspase9 activity ( ) Elongation of the hair shaft in cultured human hair follicles [94, 95] Ascorbigen Human DPC Proliferation by MTT ( ) [96] Hydrolyzed yeast extract Human DPC Acceleration of cell motility in migration by increase in the [97] number of filamentous mitochondria [98] *Clitocybin A Immortalized rat vibrissa DPC Immunopotentiator from Pantoea agglomerans 1 (IP-PA1) Proliferation by MTT ( ) Cell cycle - G0/G1 to S phase, cyclin D1, phospho-prb, phospho-cdk2 ( ) Wnt/b-catenin signaling, ERK ( ) Human DPC FGF-7, VEGF ( ) Topical and oral, Induction of hair growth [99] Bee Venom Human DPC Proliferation by WST-1 ( ) IGF-1R, VEGF, FGF-2, FGF-7 ( ) *Apo-9 0 -fucoxanthinone from Sargassum muticum Immortalized Rat vibrissadpc Proliferation by MTT ( ) VEGFR2 ( ) Wnt/b-catenin signaling, p-gsk-3b, phosphob-catenin ( ) Nuclear Translocation of Smad2/3 ( ) Topically applied, inhibition of anagen to catagen transition in C57BL/6 mice treated with Dexamethasone 5a-reductase expression in mouse skin ( ) Topically applied, anagen initiation in C57BL/6 mice Hair-fiber lengths of the rat vibrissa follicles ( ) Rat prostatic 5a-reductase activity ( ) White wax and policosanol Human DPC Proliferation by MTS ( ) Topically applied, reduction in hair loss against Testosterone treatment in Balb/c Mice, follicular length, follicular density, A/T ratio, hair bulb diameter in mice ( ) 5a-reductase activity ( ) *Undariopsis peterseniana extract Cryptomphalus aspersa Mollusc Egg Extract Phospholipids purified from Porcine Lung Tissues Immortalized Rat vibrissadpc Proliferation by MTT ( ) Cyclin D1, phospho-prb, Cyclin E, phospho-cdk2, CDK2, p-erk ( ) Wnt/b-catenin signaling, GSK-3b, catenin ( ) Human DPC Proliferation by MTT ( ) Cell migration ( ) F-actin, vimentin,vinculin, P-FAK ( ) Topically applied, anagen initiation in C57BL/6 mice Length of hair fiber length in vibrissae follicles ( ) Rat prostatic 5a-reductase activity ( ) [93] [100] [101] [102] [103] [104] Human DPC Proliferation by MTT ( ) Topically applied, initiation and progression of anagen in BALB/c and C3H mice Rice bran mineral extract Human DPC Type I collagen, ALP, fibronectin, Wnt-3a, b-catenin, versican ( ) IL-1a, TGF-b2 ( ) [105] [106] 2018 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie 435

8 Table II (continued) Compound Cells Mechanism of action in DPCs In vivo/ex vivo activity Reference Vitexin (VB1) Lignan compound Human DPC Proliferation by MTS ( ) Wnt signaling-associated genes, LEF1, Wnt5a, ALPl, versican, BMP-2, BMP-4 ( ) DKK-1, Axin2 ( ) Costunolide Human DPC Proliferation ( ) b-catenin, Gli1( ) 5a-reductase activity ( ) TGF-b1-induced phosphorylation of Smad-1/5 ( ) Growth factors and cytokines EGF Human DPC Proliferation ( ) G1 to S transition in cell cycle, ALP, IGF-1, Notch1, Jagged1, Hes1, Hes5, Survivin, Msx2 ( ) Hair shaft length in human hair follicle cultures ( ) [107] Topically applied, Improvement of hair growth [108] Growth of mink hair follicles ( ) [109] EGF, FGF Human DPC Proliferation ( ) [110] bfgf, PDGF FGF-10 Rat DPC DPCs isolated from mink skin tissues Proliferation ( ) Matrix metalloproteinase Stromelysin ( ) Proliferation by MTT ( ) Cell migration ( ) Nuclear b-catenin, SOX-9, CD133 ( ) Cell cycle (S and G2 phases) ( ) IFN-c, IL-1b, TNF-a Human DPC ICAM-1 ( ) HLA-DR against IFN-c induction ( ) [111] [112] [113] IL-1beta,IFN-gamma Human DPC PDGF ( ) [114] FGF5 Rat vibrissae DPC Proliferation ( ) [115] FGF18 Human DPC Proliferation by [3H]-thymidine incorporation ( ) [116] Angiogenin Human DPC Proliferation ( ) Elongation of hair follicles ( ) [117] Erythropoietin Human DPC Proliferation by MTT ( ) Phosphorylation of EPOR and Akt ( ) VEGF Human DPC Proliferation ( ) Phosphorylation of ERK1/2 ( ) PlGF (Placental Growth factor) Human DPC Phosphorylation of ERK, Akt, Bcl2 ( ) Cyclin D1( ) Agarose beads containing EPO implanted into dorsal skin, promotion of anagen in C57BL/6 mice Elongation of hair shafts and Ki-67 immunostaining in human follicle cultures ( ) [118] [119] Topically applied, anagen hair growth in C57BL/6 mice Hair shaft elongation in ex vivo hair organ culture ( ) PDGF-AA Human DPC Filamentous mitochondria, ATP, cell migration ( ) [121] PDGF, FGF2 Murine vibrissae DPC Proliferation ( ) [122] PDGFRa ( ) Platelet rich plasma (PRP) Human platelet lysate Mouse DPC Apoptosis by DAPI ( ) ERK, Akt, SHH ( ) Human PRP (autologous) Human DPC Proliferation by [ 3 H]-thymidine incorporation ( ) ERK, Akt, Bcl2, FGF-7, b-catenin ( ) [120] [26] Subcutaneously injected, telogen-to-anagen transition in C57BL/6 mice Human PRP Human DPC Proliferation by MTT ( ) Yield and density of hair follicles ( ) [125] Platelet-rich plasma promotes Human DPC Proliferation by MTT, ALP, Versican ( ) [126] Combination of human PRP and herbal extracts (Thymus vulgaris Urticadioica Aloe vera Rosmarinus officinalis Persea Americana Althaea officinalis Chamaemelumnobile) Human DPC Proliferation by MTT and BrdU ( ) Cyclin D1, CDK4, phosphorylation of ERK, Akt ( ) [124] [127] Porcine PRP Human DPC No effect on proliferation [128] Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie

9 Table II (continued) Compound Cells Mechanism of action in DPCs In vivo/ex vivo activity Reference 5% activated PRP as a carrier transporter for hair follicle reconstitution Placental extract *Human placental extract (alone and in combination with Minoxidil) IGF-1 isolated from Human placenta hydrolysate Human and mouse DPC Proliferation by CCK-8 ( ) Versican, b-catenin, ALP ( ) Human DPC Proliferation (BrdU) ( ) ALP, SHH, Versican, nuclear translocation of b-catenin, phosphorylation of GSK-3b, ERK ( ) TGF-b2 ( ) Human DPC Proliferation (CCK-8) ( ) b-catenin, GSK-3b ( ) Stem cells and conditioned medium Adipose tissue-derived stem Human DPC Proliferation by MTT ( ) cells pakt, perk, cell cycle (S and G2/M), Cyclic D1, CDK2 ( ) Conditioned medium of Adiposederived stem cells (ADSC-CM) Ultraviolet B Preconditioned- ADSC-CM Conditioned medium (CM) from Keratinocyte stem/progenitor cells (KSCs) Human dermal stem/progenitor cell-derived conditioned medium Neural Stem Cells extract and conditioned medium Extracellular vesicles derived from MSCs Peptide/proteins Tripeptide-copper complex L- alanyl-l-histidyl-l-lysine-cu2+ (AHK-Cu) PRP scaffold constructs containing mouse trichogenic cells grafted in nude mice, promotion of hair growth, Promotion of mouse hair follicle formation in vivo Root hair elongation in rat vibrissa hair follicles, delay in catagen progression, TGFb2 ( ) Subcutaneously injected, promotion of hair growth in C57BL/6 mice, number and size of hair follicles ( ) Topical application and Subcutaneously injected in C(3)H/NeH mice, earlier conversion of telogen to anagen Elongation of human hair follicles ( ) Human DPC Proliferation by CCK-8 ( ) Subcutaneously injected, induced the anagen phase and increased hair regeneration in C(3)H/NeH mice Human DPC Proliferation by MTT ( ) Subcutaneously injected, telogen-to-anagen transition upon injection in C(3)H/HeN mice Elongation of hair shaft in ex vivo human follicle culture Human DPC Proliferation by CCK-8 ( ) Cell cycle (S phase), pakt, perk1/2 ( ) Subcutaneously injected, earlier conversion of telogen to anagen in C3H/HeN mice Elongation of hair shaft length human hair organ culture Human DPC ALP, SOX2, S100ß, versican, CORIN, Wnt3a ( ) Topical application, hair regeneration in C57BL/6 mice [136] Immortalized human DPC Proliferation by WST-1 ( ) IGF-1, HGF, KGF, VEGF ( ) Modulation of TGF-b2, BMP-2, BMP-4, BMP-6 Human DPC Proliferation by CCK-8 ( ) pakt, perk1/2, Bcl2, VEGF, IGF-1, cell migration ( ) Human DPC Proliferation by MTT, Bcl2/Bax ( ) Apoptosis (annexin, caspase-3), PARP ( ) [129] [130] [131] [132] [133] [134] [135] Topical application, promotion of hair growth in C57BL/6 mice [137] Intradermal injection, conversion from telogen to anagen in C57BL/6 mice. Wnt3a, Wnt5a and versican ( ) [138] Elongation of human hair follicles ( ) [139] Tetrapeptide AcSDKP Human DPC Proliferation ( ) Hair shaft elongation in the ex vivo-cultured hair follicles ( ) [140] [141] Aspartate-serine-serine (8DSS) peptides Human DPC Wnt10b, LEF-1, c-myc, nuclear b-catenin, phosphorylation of GSK3b, Akt ( ) Topically applied, induction of early telogen-to-anagen transition inc57bl/6 mice, b-catenin ( ) Lysozyme DPC ALP, LEF 1 ( ) Hair shaft elongation in mouse vibrissae follicle culture ( ) [142] [143] Oleuropein Human DPC Proliferation by MTT ( ) LEF1, Cyc-D1, b-catenin ( ) Sphinganine in Combination with Caffeine or Creatine Mimetics Biomimetic peptide Sh- Polypeptide 9 (CG-VEGF) Topically applied, anagen-phase induction in C57BL/6N mice Wnt10b, FZDR1, LRP5, LEF1, Cyc-D1, IGF-1, KGF, HGF, VEGF and b-catenin protein ( ) Human DPC Proliferation by MTT ( ) [144] Human DPC Survival, proliferation and tubulogenesis of human microvascular endothelial cells (HMVEC) (total tubule length and number of junctions) ( ) b-catenin ( ) [145] Ceramide mimetic Human DPC Sphinganine, sphingosine, ceramides ( ) Promotion of hair growth in ex vivo cultured human hair follicles, earlier conversion of telogen into anagen [146] 2018 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie 437

10 Table II (continued) Compound Cells Mechanism of action in DPCs In vivo/ex vivo activity Reference Lipid nanocarrier Squarticles containing diphencyprone (DPCP) and minoxidil Anti-PDGF receptor b antibodyconjugated squarticles Human DPC VEGF ( ) [147] Human DPC Proliferation by MTT ( ) VEGF ( ) Light Laser diode light irradiation Human DPC Proliferation ( ) Photobiomodulation therapy with light-emitting diodes [148] [149] SOX-2, FGF-7 ( ) Human DPC Proliferation by MTT ( ) [150] Light-Emitting Diode Light Human DPC Proliferation by MTT ( ) Wnt3a, Wnt5a, Wnt10b, b-catenin, axin-2, cyclin D1, phosphorylation of MEK, JNK, ERK ( ) Elongation of human hair follicles ex vivo ( ) [151] Electromagnetic field 1,763 MHz radiofrequency Human DPC VEGF, IGF-1, Bcl2, CCND1, p-mapk-1 ( ) Hair shaft elongation in ex vivo human hair organ cultures, Ki67 ( ) TUNEL ( ) Extremely low frequency electromagnetic field Human DPC Proliferation by MTT ( ) MAP-2 ( ) [153] Electrical stimulation Alternating current Human DPC Proliferation by MTT Wnt/b-catenin, Ki67, p-erk and p-akt ( ) Electrical Stimulation in combination with Minoxidil Human DPC Proliferation (MTT) ( ) Ki-67, b-catenin, Wnt/b-catenin ( ) Others Retinol Human DPC Proliferation by MTT, Bcl-2/Bax ratio, phosphorylated ERK and Akt ( ) Sinapic acid Human DPC Proliferation ( ) Activation of Akt, IGF-1, VEGF, p-gsk-3b, b-catenin, cell cycle progression ( ) Arachidonic acid Human DPC Proliferation by MTT ( ) FGF-7, FGF-10, HGF, perk, pakt, pcreb, Bcl2 ( ) *Valproic acid Human DPC ALP, Wnt/b-catenin, BMP4 ( ) Proliferation, b-catenin, p- GSK-3b ( ) Topically applied, increased hair growth in rabbits, PDGF, SOX9, EGF and IGF ( ) [152] [154] [155] Hair follicle length ( ) [27] [156] Topically applied, prolongation of anagen phase in telogen-stage C57BL/6 mice Hair shaft elongation in hair follicle culture ( ) Topically applied, hair re-growth in male C3H mice Filaggrin, loricrin, ALP, ERK and Akt ( ) Mycophenolic acid Human DPC Proliferation, Axin2, p-erk, Bcl-2/Bax ( ) Topically applied, anagen induction in C57BL/6 mice [160] Aggrecan, biglycan, fibronectin, Human DPC Proliferation by Ki67 ( ) [161] hyaluronic acid (HA) GSK-3 inhibitor, (2 0 Z,3 0 E)-6- Human DPC ALP, IGF-1, b-catenin ( ) [162] bromoindirubin-3 0 -oxime (BIO) [163] Cilostazol Human DPC Proliferation by BrdU ( ) IGFBP-1, IGFBP-3, IGF-2, M-CSF, M-CSFR, PDGF-Rb, PDGF-AB, PDGF-BB ( ) 3-Deoxysappanchalcone, Tofacitinib Bimatoprost Immortalized rat vibrissa DPC Human DPC Proliferation (xcelligence system) T cell factor, STAT3, CDK4, FGF, VEGF, Phosphorylation of b-catenin ( ) Proliferation, CDK2 and Cyclin E b-catenin, Cox-2 ( ) cell cycle (subg1 population) ( ) Topically applied, promotion of anagen phase in C57BL/6 mice by stimulating vessel formation, phosphorylated kinase, c-junnterminal kinase, P38 ( ) Shaft elongation in human hair follicle culture ( ) Topically applied, promotion of the anagen phase of hair growth in C57BL/6 mice [157] [158, 159] [164] Hair-fiber length of rat vibrissa follicles ( ) [165] Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie

11 Table II (continued) Compound Cells Mechanism of action in DPCs In vivo/ex vivo activity Reference [166] Adenine Human DPC Proliferation by cell counting, AMPK, NAD+ levels ( ) Prevention of decrease in gene expression - CNB1, CCNB2, CCNE1, ASPM, PKB, SPAG5, CDC25C, PCNAand TERT, b-galactosidase activity ( ) Adenosine Human DPC FGF-7, camp ( ) Elongation in human scalp hair follicle organ cultures( ) [167] [168] Activated and elongated the anagen phase mouse vibrissae hair follicles Human DPC Proliferation by 3H-thymidine incorporation, b-catenin, FGF-7, FGF-2, IGF-1, and VEGF, perk, pcreb and pakt ( ) [169] Estradiol (E2) Human DPCs cell lines ALPL ( ) APOE, BMP4, GSK3b, LEF1, LRP4, MSX2, NOG, VCAN, WIF1 ( ) Wnt5a ( ) Angiopoietin 2 ( ) ( ) Stimulation, ( ) Inhibition, *Minoxidil was included as positive control for all the parameters. Other agents In addition, various other compounds have demonstrated stimulatory effect on DPCs at molecular level [27, ]. Protective effect in hair growth against stress Various hair growth promoting agents have shown stimulatory effects on DPCs in response to stress inducing conditions that suggests their efficacious effect in hair loss triggered by environmental damage (Table III). H 2 O 2 induced stress Oxidative stress results in the damage of DNA, proteins and lipids resulting in graying and hair loss. Treatment of DPCs with H 2 O 2 results in loss of viability, reduction of cells in G1 phase of cell cycle and modulation of mirna genes involved in growth arrest and cell death [170]. Arctiin and Trihydroxyethylrutoside (troxerutin) have demonstrated protection against H 2 O 2 induced damage in DPCs [171,172]. UV induced stress Ultraviolet (UV) radiation impairs the intracellular functions by inducing DNA damage, generation of ROS and repression of growth and cycling of hair follicles [173]. Synergistic combinations of b-glucogallin and gallates, concentrate of liquid endosperm of Cocos nucifera and selenopeptides showed protective effect against UVB in DPCs [174]. DHT induced stress Androgen-driven changes in the autocrine and paracrine factors produced by the scalp DPCs may play an essential role in androgenpotentiated balding. Dihydrotestosterone (DHT) is known to induce androgenic alopecia by shortening the hair follicle growth phase, resulting in hair loss. DHT damages DPCs by upregulation of Dickkopf 1 (DKK-1), induction of cell death, increase in ROS and senescence in DPCs [175, 176].A variety of compounds have demonstrated protective effect against DHT induced stress in DPCs [ ]. Cortisol induced stress Cortisol is a stress-related neurohormone of the hypothalamic-pituitary-adrenal (HPA) axis, which inhibits hair growth. A selective inhibitor of 11b-Hydroxysteroid Dehydrogenase Type 1, attenuated the anti-proliferative effects of Cortisol in DPCs [185]. Dexamethasone induced stress Glucocorticoids (GC) are synthesized mostly in the adrenal gland in response to stressful conditions, and mediate cellular damage such as hair loss. Treatment of DPCs with Dexamethasone resulted in decreased cell viability, reduced VEGF and HGF secretion, ER alpha levels and increased expression of aromatase mrna [186, 187]. Geranium sibiricum extract alleviated the damaging effects in DPCs induced by Dexamethasone [188]. IFN-c-induced stress IFN-c induces catagen in hair follicles by reducing b-catenin, activating GSK-3b, enhancing the expression of DKK-1 and 2018 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie 439

12 Table III Protective effects of various agents on DPCs against stress conditions Compound Cells Mechanism of action in DPCs In vivo/ex vivo activity Reference H 2 O 2 induced stress Arctiin, active lignin isolated from Arctium lappa Human DPC H 2 O 2 -induced loss of cell viability by WST-1 ( ) sub-g1 phase accumulation and G2 cell cycle arrest, ROS levels ( ) SA-b-gal activity ( ) mirna - MAPK, Wnt signaling [171] Troxerutin Human DPC Cell survival against H 2 O 2 by WST-1 ( ) [172] Cell cycle-subg1 cell population ( ) ROS ( ) SA-b-gal activity ( ) mirna- MAPK, Wnt signaling UV induced stress Seleneopeptides Human DPC Cell survival by NRU against UVB ( ) [174] DHT induced stress CM from Rat bone marrow mesenchymal stem cultured in Wnt1a virus-containing medium Human DPC LEF-1, Versican, Ptc-1, Gli-1, cell migration against DHT ( ) Intradermal injection, telogen to anagen transition inc57bl/6 mice LEF-1, Versican, Ptc-1, Gli-1, ALP ( ) Scutellaria baicalensis extract Human DPC Nuclear translocation of the androgen receptor stimulated by DHT [178] ( ) Proliferation ( ) Forsythiaside-A Human DPC Apoptosis induced by DHT ( ) Inhibited entry into catagen induced by DHT, an increase in mouse hair density and thickness TGF-b2, caspase-3 and 9 ( ) Bcl2/Bax ( ) [179] Red ginseng extract Human DPC Proliferation by (CCK-8), p-erk, p-akt ( ) DHT induced upregulation of AR mrna ( ) Ginsenoside F2 Human DPC Proliferation by MTT, Bcl2/Bax against DHT ( ) TGF-b2, sp1, Smad2, Smad3, SCAP, SREBP, caspase-12 against DHT ( ) EGCG Human DPC DHT-mediated cell death ( ) Intracellular ROS levels, senescence, cell cycle SubG1 cell population ( ) [177] Subcutaneously injected, [180] promoted hair growth in C57BL/ 6 mice. Orally administered, hair growth [181] promotion in C57BL/6 mice treated with DHT, hair cell apoptosis and premature entry to catagen ( ) [182] L-ascorbic acid 2-phosphate Human DPC DHT induced DKK-1 ( ) [183] L-threonate Human DPC DHT-induced DKK-1 ( ) [184] Cortisol induced stress 11b-Hydroxysteroid Dehydrogenase Type 1 inhibitor Human DPC Anti-proliferative effects of cortisol by Ki67 staining ( ) Reversal of cortisol-induced suppression of Wnt5a, ALP, and VEGF ( ) Dexamethasone induced stress Geranium sibiricum L extract Human DPC Proliferation (MTT), cell migration, Ki-67 positive cells, HGF against Dexamethasone ( ) TGF-b1 ( ) IFN-c-induced stress *Mycophenolate Human DPC Proliferation (MTT), b-catenin, ALP, axin2, IGF-1, FGF 7, FGF10 against IFN-c ( ) GSK3b, DKK-1, TGF-b2 ( ) Cisplatin induced stress Phyllanthus emblica Linn (emblica) extracts Human DPC Cell viability by MTT ( ) ROS induced by Cisplatin ( ) [185] [188] Stimulation of growth of IFN-ctreated human hair follicles [189] [191] ( ) Stimulation, ( ) Inhibition, *Minoxidil was included as positive control for all the parameters. catagen inducer transforming growth factor (TGF)-b2. Mycophenolate (MPA) is an immunosuppressant drug, which showed protective effect against IFN-c-induced stress conditions in DPCs [189]. Cisplatin induced stress Chemotherapy-induced alopecia (CIA) involves massive apoptosis of hair follicle cells. Cisplatin is a chemotherapeutic drug, which Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie

13 Table IV Inhibitory effects of various agents on DPCs Compound Cells Mechanism of action in DPCs In vivo/ex vivo activity Reference Testosterone/DHT Testosterone, Dihydrotestosterone Human DPC Proliferation by 3H-thymidine ( ) [194] Testosterone Macaque Supervillin (F-actin-binding protein) ( ) [195] DPC isolated from bald frontal scalp Testosterone and 5adihydrotestosterone Human DPC Nitric oxide, inducible nitric oxide synthase ( ) [196] (DHT) DHT Human DPC TGF-b2, activation of caspase network ( ) [197, 198] Wnt5a and Wnt10b mrna ( ) Wnt antagonist Dkk-1 ( ) DHT Rat vibrissae DPC No effect on proliferation (by MTT) Cell cycle arrest -G1 cell population, p27(kip1), Phosphorylation of Smad2/3, HSP27, p38, mtor, ( ) Cyclin E, cyclin D1, CDK2, b-catenin, Cox-2, NF-jB p65 ( ) [199] Stanozolol (Testosterone analogue) Synthetic androgen, methyltrienolone (R1881) Vitamin D3 1,25-dihydroxyvitamin D3 (1,25 (OH)2D3) Peptides Calcitonin gene-related peptide (CGRP) Human DPC Rat vibrissae DPC Human DPC Modulation of 13 genes FasL, Caspase 8 and Bcl-2, AR, ER, IGF1R, GHR, FGF2, FGF7, SRD5A1, SRD5A2, UBQ, b- actin [200] ROS, TGF-b1 ( ) [201] ALP, TGF-b2 ( ) Proliferation by BrdU ( ) Apoptosis(annexin) ( ) Wnt10b, ALPL, TGF-b2 ( ) [202, 203] Human DPC IL-1b ( ) Shortening of the anagen stage of the hair cycle in cultured hair follicles [204] Human DPC Cell proliferation, ALP ( ) [205] CXXC-type zinc finger protein 5 (CXXC5) Corticotropin-Releasing Factor Human DPC Modulation of KGF, Wnt5a, TGF-beta2, Nexin camp, proopiomelanocortin (POMC) ( ) CRF receptors ( ) Growth factors/cytokines bfgf, VEGF Connective tissue growth factor (CTGF) Rat vibrissae DPC Human DPC Inhibition of hair shaft elongation in cultured human hair follicles Collagen ( ) [207] Proliferation ( ) ALP, b-catenin, BMP2, TGF-b2( ) Versican, a-smooth muscle actin (SMA) ( ) [206] [208] IL-1-a Human DPC NF-kB pathway, KGF, VEGF, IL-8, GMCSF, Nexin-1( ) [209] Others PMA Human DPC PKC-a,, c ( ) [210] Stress serum Rat and human DPC Viability by MTT ( ) Cell cycle arrest in G1 phase ( ) [211] 6-Ginegrol Human DPC Proliferation by MTT ( ) Apoptosis (annexin) ( ) Bcl2/Bax ( ) Topical application, delay in anagen induction from telogen in C57BL/6 mice Inhibition of hair shaft elongation in cultured human hair follicles Colchicine Rat Proliferation, ALP activity, IjBa phosphorylation ( ) [213, 214] vibrissae DPC Taxol Rat Apoptosis - caspase 3 activation DNA fragmentation ( ) [215] vibrissae DPC Modulation of 11 proteins involved in Ca2+ dependent transport Retinoic acid, glucocorticoids, Human DPC IGFBP-3 ( ) [216] IGF-1 15-deoxy prostaglandin J2 Human DPC Viability ( ) Apoptosis in cultured human hair [217] Apoptosis ( ) follicles Prostaglandin D2 (PGD2) Human DPC AR expression and Akt signaling through prostaglandin D2 receptor 2 ( ) TGFb1, CREB, LEF1, IGF-1, GSK3b ( ) [218] [212] ( ) Stimulation, ( ) Inhibition Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie 441